Efficient synthesis of CRISPR-Cas13a-antimicrobial capsids against MRSA facilitated by silent mutation incorporation

In response to the escalating global threat of antimicrobial resistance, our laboratory has established a phagemid packaging system for the generation of CRISPR-Cas13a-antimicrobial capsids targeting methicillin-resistant Staphylococcus aureus (MRSA). However, a significant challenge arose during the packaging process: the unintentional production of wild-type phages alongside the antimicrobial capsids. To address this issue, the phagemid packaging system was optimized by strategically incorporated silent mutations. This approach effectively minimized contamination risks without compromising packaging efficiency. The study identified the indispensable role of phage packaging genes, particularly terL-terS, in efficient phagemid packaging. Additionally, the elimination of homologous sequences between the phagemid and wild-type phage genome was crucial in preventing wild-type phage contamination. The optimized phagemid-LSAB(mosaic) demonstrated sequence-specific killing, efficiently eliminating MRSA strains carrying target antibiotic-resistant genes. While acknowledging the need for further exploration across bacterial species and in vivo validation, this refined phagemid packaging system offers a valuable advancement in the development of CRISPR-Cas13a-based antimicrobials, shedding light on potential solutions in the ongoing battle against bacterial infections.

The phage-specific packaging site (pac), encoding signals for packaging phagemid into phage capsids to generate AB-capsids, comprises four genes: terL, terS, rinA, and rinB.We assessed the impact of knocking out pac genes from lysogenized Tan2 prophage on the efficiencies of phagemid packaging into phage capsids (Fig. 1A).In our previous study, the generation of AB-capsids using phage-lysogenized S. aureus strain with terL and terS deletion (RN4220 ΦTan2ΔterL/terS ) resulted in a relatively higher yield and lower levels of contamination with wild-type phages (manuscript in preparation).Here, we cloned different combinations of pac genes (terS, terL-terS, terL-terS-rinA, or terL-terS-rinA-rinB) onto phagemids and evaluated their efficacy in AB-capsid generation and preventing wildtype phage contamination when using RN4220 ΦTan2ΔterL/terS as the host cell (Fig. 1B).In brief, each phagemid was transformed into RN4220 ΦTan2ΔterL/terS , and the transformants were chemically-induced with mitomycin C (MMC) to activate prophages.This in turn initiated phage synthetic processes resulting in the generation of AB-capsids (packaging of phagemids) and/or wild-type phages (packaging of phage DNA).As shown by the red bars in Fig. 1C, the three phagemids carrying the gene combinations of terL-terS, terL-terS-rinA, and terL-terS-rinA-rinB were packaged, generating AB-capsids with titers of 6.33 ± 1.89 × 10 5 , 5.17 ± 1.19 × 10 7 , and 1.53 ± 0.99 × 10 8 TFU/ ml, respectively.However, wild-type phages were also produced in the case of phagemids carrying terL-terS-rinA and terL-terS-rinA-rinB (blue bars), at approximately 4.00 ± 2.16 × 10 5 and 1.27 ± 0.66 × 10 6 PFU/ml, respectively.Notably, in the case of the phagemid carrying only terL-terS, no contamination with wild-type phages was observed; however, there was a significant reduction in AB-capsids yield.Furthermore, phagemids carrying only the terS packaging gene showed neither AB-capsids nor wild-type phage detection.This suggests that pac genes, especially terL-terS, are essential, while rinA and rinB are needed for efficient packaging of phagemid into the phage capsid.

Removal of homologous sequences between prophage and phagemid
Upon learning that the entire pac genes are necessary for efficient packaging of the phagemid but lead to wildtype phage contamination, we hypothesized that the overlapping homologous region between prophage and phagemid might be a crucial contributing factor.To address this, we deleted the 57 bp long homologous sequence existing between the phagemid and Tan2 prophage in the host cell RN4220 ΦTan2ΔterL/terS (Fig. 1B), aiming to enhance the generation of AB-capsids while minimizing wild-type phage contaminations.
Initially, we generated a deletion mutant, RN4220 ΦTan2ΔterL1/terS , by removing the 57 bp homologous sequence at the 3ʹ end of the terL gene in the prophage of RN4220 ΦTan2ΔterL/terS (Fig. 2A).We then transformed the mutant host cell, along with the original host cell RN4220 ΦTan2ΔterL/terS , with phagemid-LSAB to produce AB-capsid.Subsequently, the production of both AB-capsids and wild-type phages was compared.Unexpectedly, the results demonstrated a significant decrease in the production of both AB-capsids and wild-type phages, from 7.87 ± 1.64 × 10 5 to 5.33 ± 2.49 × 10 1 TFU/ml and 9.33 ± 7.54 × 10 2 to 6.00 ± 5.89 × 10 1 PFU/ml, respectively (Fig. 2B).This finding Figure 1.Influence of packaging genes on phagemid packaging and AB-capsid generation.(A) Schematic representation of phagemid packaging and AB-capsid generation.S. aureus cells with an integrated prophage Tan2 in their chromosome are transformed with the phagemid.The phagemid carries the CRISPR-Cas13a system, chloramphenicol resistance gene, tetracycline resistance gene, ori of S. aureus and E. coli, and the packaging gene(s).After transformation, mitomycin C induction activates phage synthetic processes, initiating the translation of phage structural proteins and leading to phage assembly.The phagemid is loaded into capsids, as it carries packaging signals that can be recognized during phage packaging machinery, thereby yielding AB-capsids.(B) Illustration depicting the packaging genes carried on the prophage genome of host S. aureus strain RN4220 ΦTan2ΔterL/terS and the phagemids.The upper panel shows the genome of the prophage integrated in host strain RN4220 ΦTan2ΔterL/terS , with gray arrows indicating the knocked-out genes of terL and terS.The blue arrows represent the packaging gene(s) of the phagemids used in this experiment.The phagemid-LSAB, phagemid-LSA, phagemid-LS and phagemid-S corresponded respectively to phagemid pLK19::TerL-TerS-RinA-RinB, pLK19::TerL-TerS-RinA, pLK19::TerL-TerS, and pLK19::TerS, in our previous study (manuscript in preparation).The light red and light blue areas indicate homologous regions between prophage and phagemids.(C) Quantifications of transduced colony-forming unit (TFU) of AB-capsids and plaque forming unit (PFU) of wild-type phage packaged using host cell RN4220 ΦTan2ΔterL/terS transformed with any of the four phagemids (n = 3).Red bars indicate log TFU/ml, and blue bars represent log PFU/ml.Error bars represent standard deviations.N.D. not detected.
indicates that the deletion of the 57 bp homologous sequence at the 3ʹ end of the terL gene is not viable for ensuring the efficient packaging of the phagemid-LSAB.

Modification of the homologous sequences on phagemids
Deletion of the 57 bp homologous region shared between phagemid-LSAB and the prophage in RN4220 ΦTan2ΔterL/terS is not plausible considering that this 57 bp sequence is situated within the overlap region of the end of terL and the beginning of a neighboring gene that encodes a portal protein.The deletion of this 57 bp region resulted in the removal of the initial codon of the portal gene, rendering it dysfunctional.As an alternative way to eliminate sequence homology between prophage and phagemids, we introduced silent mutations into the 57 bp homologous region of terL, replacing 24 nucleotides without causing amino acid substitutions.This modification reduced nucleic acid sequence identity to about 58%, resulting in the creation of phagemid-LSAB(mosaic) (Fig. 3A, Table S4).Additionally, the phagemid-ΔLSAB(cut) with a deletion of the 57 bp sequence was prepared as a control, other than phagemid-LSAB.Using these three phagemids, we compared their efficacy in the production of AB-capsids and prevention of wild-type phage contamination.Surprisingly, as depicted in Fig. 3B and C, phagemid-LSAB(mosaic) yielded 6.53 ± 0.61 × 10 5 TFU/ml of pure AB-capsids with undetectable levels of wild-type phage contamination.The AB-capsids titer showed no significant difference compared to that produced by phagemid-LSAB (1.51 ± 0.35 × 10 6 TFU/ml).As anticipated, phagemid-ΔLSAB(cut) generated neither AB-capsids nor wild-type phage.

Sequence-specific killing of phagemid-LSAB(mosaic)-based AB-capsids against S. aureus
Considering the above discoveries, our focus shifted towards exploring the sequence-specific killing capabilities of the generated AB-capsids against S. aureus by targeting various important/frequently-encountered antibiotic resistant genes to address clinical needs.The phagemid-LSAB(mosaic) harbors the chloramphenicol resistance (CpR) gene cpr from pIMAY, which functions in both S. aureus and E. coli 10 .Additionally, it carries the tetracycline resistance (TetR) gene tetM from SaPIbov2 bap::tet30 11 .The inclusion of double antibiotic resistance markers eases the screening of AB-capsids killing activities across a wide spectrum of clinical S. aureus strains, considering that the 500 clinical isolates examined rarely possess both genes simultaneously.In addition to the mecA gene, we selected the following eight antibiotic-resistant genes of S. aureus as preliminary target genes to test the sequence-specific bacterial killing effect of AB-capsids against antibiotic-resistant S. aureus: aph(2ʹ), aadD, aph(3ʹ), aac(6ʹ), ermB, fusC, mphC, and tetK (Table S1).Importantly, aph(2ʹ), aph(3ʹ), and aac(6ʹ) are associated with gentamicin and tobramycin resistance (aminoglycoside antibiotics ); aadD with streptomycin resistance (aminoglycoside antibiotics); ermB with erythromycin resistance (macrolide antibiotics); fusC with fusidic acid resistance; mphC with resistance towards macrolides; and tetK towards tetracyclines.
In each instance, 25 bp long spacers complementing each of the aforementioned genes were designed and inserted into the phagemid to generate a resistance gene-targeting phagemid-LSAB(mosaic) series.The non-targeting phagemid-LSAB(mosaic) without a spacer (Non-T) serves as a negative control.Subsequently, each phagemid was transformed into RN4220 ΦTan2ΔterL/terS to produce AB-capsids targeting the specified antibiotic-resistant genes mentioned above.Sequence-specific bactericidal activity of the resultant AB-capsids were then evaluated using spot assay (Fig. 4A).Notably, all nine different AB-capsids exhibited pronounced bactericidal activity against their corresponding target S. aureus strains.These strains include both the transformants of RN4220, where each individual target gene was over-expressed on plasmid pKAK (Fig. 4B), and clinical isolates JMUB1278 and JMUB3007, which carry antibiotic-resistant genes aac(6ʹ) and mecA (Fig. 4C), and aadD and mecA (Fig. 4D), respectively.The specified antibiotic-resistant genes targeted by the generated AB-capsids are highlighted in red.

Discussion
Antimicrobial resistance has emerged as a global health threat, partly due to the stagnation in antibiotic development.To tackle this issue, there has been a renewed interest in phage therapy, coupled with innovative strategies like phage-antibiotic combinations, phage-derived enzymes, and phage bioengineering 12,13 .The application of natural or engineered phages, either alone or in combination with traditional antibiotics, in compassionate therapies or clinical trials targeting infections with Acinetobacter baumannii 14 , Mycobacterium abscessus 15 , S. aureus 16 , and Pseudomonas aeruginosa 17 further supports the feasibility of phage therapy in clinical settings.In our laboratory, we have developed AB-capsids, a novel non-traditional antibacterial agent designed to combat the multidrug-resistant MRSA strains 5 .These AB-capsids are non-replicative phage particles loaded with mecA-targeting CRISPR-Cas13a through our established phagemid packaging system, demonstrating promising in vitro results.However, the translation from controlled laboratory environment to the dynamic realm of clinical applications poses substantial challenges.The shift from experimental setup to clinical application necessitates careful consideration of several critical factors before deploying natural or engineered phages.These factors include: (1) specificity and efficiency in eradicating pathogenic bacteria, (2) ease of preparation to achieve high titer and purity, (3) stable storage without substantial deterioration in bactericidal activity, and (4) safety concerns, ensuring the absence of endotoxins or harmful contaminants and preventing undesirable horizontal gene transfer 18 While the phagemid packaging system offers a promising avenue for the efficient generation of AB-capsids, this current production system faces issues such as contamination with wild-type phages and low AB-capsid titers, necessitating refinement to enhance efficiency and reliability.Addressing these challenges is therefore essential to advance the clinical potential of AB-capsids.
In this study, AB-capsids were generated using RN4220 ΦTan2ΔterL/terS , a phage-lysogenized S. aureus strain with terL and terS deletion, to minimize the risk of wild-type phage contamination.The TerS-TerL complexes, crucial components in phage DNA packaging 19 , were identified as pivotal in this process.It has been documented that the complete elimination of natural phage production can be achieved through the deletion of prophage terS 20 .However, despite these measures, wild-type phage contamination was observed during the packaging of phagemid-LSAB and phagemid-LSA using RN4220 ΦTan2ΔterL/terS .This phenomenon was traced back to the presence of homologous regions, both at the 5ʹ end (rinA or rinA-rinB) and 3ʹ end (57 bp sequence) of phageencoded packaging site genes, shared between phagemids and prophage (Figs.1B, 2A).It is postulated that these homologous sequences compensate for the loss of terS and terL on the prophage, facilitating the concatemerization of natural phage DNA 21,22 and subsequent packaging of natural phages.As evidenced in the packaging of phagemid-LS, the removal of rinA-rinB successfully abolished 5ʹ end homology, consequently eliminating wild-type phage contamination.However, the absence of rinA adversely affected AB-capsid production (Fig. 1C).Ferrer et al. previously reported that rinA plays a crucial role as a regulator of two major gene clusters of phages, specifically the morphogenesis and lysis clusters 23 .Deletion of rinA resulted in a significant reduction in phage/ phage-like particles.Given these findings, the presence of rinA on phagemids is believed to be advantageous in our phagemid packaging system, contributing to the optimal production of AB-capsids.www.nature.com/scientificreports/ As an alternative strategy to address wild-type phage contamination, a 57 bp homologous sequence on the 3ʹ end of terL gene in the prophage of RN4220 ΦTan2ΔterL/terS was removed.The deletion of the 3ʹ end homologous region on the prophage proved detrimental, resulting in a significant reduction in AB-capsid titers from 7.8 × 10 5 TFU/ml to 5.33 × 10 2 TFU/ml (Fig. 2B).Notably, the 3ʹ end homologous region encompasses the initial codon of a gene encoding the phage portal protein (Figs.1B, 2A).The portal protein serves as the docking subunit for TerL, initiating phage DNA packaging 24 .The interaction between TerL and the portal protein is crucial for the assembly of phage capsids, particularly in tailed phages 25 .On the other hand, removal of 3ʹ end homologous region from phagemids is undesirable, considering the importance of C-terminal of terL in cleaving phage DNA during packaging [26][27][28] .Moreover, while controversial, the C-terminal of terL is proposed to be the domain that binds to portal protein to initiate phage DNA packaging due to its closer proximity to the portal compared to its N-terminal ends 29 .Complete elimination of the 3ʹ end homologous region, whether from the prophage or phagemids, is thus deemed inappropriate (Figs. 2, 3).
Subsequently, we employed a strategy involving the introduction of silent mutations into the 3ʹ end homologous region on phagemids.Forty-two silent mutations were intricately incorporated into the C-terminal of terL, replacing 24 nucleotides without inducing amino acid substitutions.This modification resulted in the creation of a phagemid termed phagemid-LSAB(mosaic) (Fig. 3A, Table S4), which facilitated the production of high titers of AB-capsids while completely eliminating wild-type phage contamination (Fig. 3B).Notably, this highlighted that a reduction in sequence similarity at the 57 bp region between prophage and phagemids to approximately 58% proved sufficient to impede recombination between the two DNA sequences and hinder natural phage production.These results indicated that we successfully optimized the phagemid packaging system, addressing the complex interaction between the packaging site on the phagemid and the prophage in host cells.The AB-capsids generated from RN4220 ΦTan2ΔterL/terS using phagemid-LSAB(mosaic) were subsequently subjected to testing against S. aureus strains carrying targeted antibiotic-resistant genes (Fig. 4A).The applicability of phagemid-LSAB(mosaic)-based AB-capsids was not merely confined to the laboratory-derived strains, instead their sequence-specific killing efficacy were also demonstrated using clinical isolates of S. aureus strains carrying different target antibiotic-resistant genes (Fig. 4B, C).While acknowledging the need for further exploration into crucial aspects such as stability and safety, this study presented a refined phagemid packaging system effective in generating phage-based antibacterials with improved titers and purity, marking a significant stride toward realizing the potential of phage therapy.
While this study marks a significant advancement in targeted antimicrobial agents development, it is crucial to acknowledge its inherent limitations.The specificity of the AB-capsids, primarily demonstrated against MRSA, raises questions about its generalizability to diverse bacterial species.The complexity of bacterial strains and their potential variations in response to the optimized system necessitate thorough examination across multiple strains.Additionally, the lack of in vivo validation poses a notable limitation.While in vitro experiments lay the groundwork for understanding the system's performance, translating these findings into relevant animal models or clinical settings is essential.In vivo validation would provide insights into the practicality and effectiveness of the system in complex biological environments, and address concerns about potential off-target effects.Despite improvements in titers and purity, future research addressing stability and safety concerns associated with the phagemid packaging system is necessary.
In conclusion, the optimization of the phagemid packaging system through the incorporation of silent mutations represents a significant advancement in the development of CRISPR-Cas13a-antimicrobial capsids against MRSA.The study's findings not only contribute valuable insights into combating antibiotic resistance but also pave the way for future research exploring the diverse applications and potential collaborations in the dynamic field of CRISPR-based antimicrobials.While the study provides profound insights, recognizing and addressing its limitations through continued research and refinement is paramount for unlocking the full translational potential of these findings.

Bacterial strains and culture conditions
S. aureus strains were cultivated at 37 °C in tryptic soy broth (TSB; BD Difco, USA) or on tryptic soy agar (TSA; BD Difco, USA).Chloramphenicol (Cp) at a concentration of 10 µg/ml, and/or Tetracycline (Tet) at 5 µg/ml was added when necessary.E. coli strains were grown at 37 °C in Luria-Bertani (LB) broth (BD Difco, USA) or on LB agar (BD Difco, USA), with the addition of following antibiotics when appropriate: 30 µg/ml of kanamycin (Km), 100 µg/ml of ampicillin (Amp), 5 µg/ml of Tet and/or 10 µg/ml of Cp.All S. aureus and E. coli strains used in this study were listed in Supplementary Table S1 10,30 .
Construction of pac deletion mutants S. aureus RN4220 ΦTan2ΔterL/terS and RN4220 ΦTan2ΔterL1/terS 0The RN4220 ΦTan2ΔterL/terS and the RN4220 ΦTan2ΔterL1/terS mutants were generated through allelic exchange using temperature-sensitive plasmids.The RN4220 ΦTan2ΔterL/terS mutant, with deletions of the terL and terS genes from Tan2 prophage, was generated with the plasmid pIMAY 10 , as described in our previous study (manuscript in preparation).In this study, we aimed to generate another mutant derivative of RN4220 ΦTan2 WT , RN4220 ΦTan2ΔterL1/terS .This new mutant has a deletion of a 57 bp region at the 3ʹ end of the terL gene.We generated this mutant using the plasmid pKAK 31 through allelic exchange.For the construction of RN4220 ΦTan2ΔterL1/terS , 500-1000 bp upstream and downstream flanking sequences of terL-terS of prophage Tan2 were amplified with two different primer sets: pKFT KO SL no promoter S1 v2/pKFT KO SL no promoter AS1 v2 and pKFT KO SL no promoter S2 v2/pKFT KO SL single and no promoter AS2, employing KOD FX Neo polymerase (Toyobo, Japan).The pKFT 31 plasmid was also PCR-amplified using the same polymerase with primer set pKFT MCS outside S/pKFT MCS outside AS.The three PCR-amplified fragments were subsequently ligated using NEBuilder ® HiFi DNA Assembly (New www.nature.com/scientificreports/England Biolabs, USA) to ultimately generate pKFT_ΔTerL/TerS knockout plasmid, which was then transformed into E. coli DC10B and selected on LB agar supplemented with 100 µg/ml of Amp.The constructed plasmid was extracted and verified by Sanger sequencing.Following that, sequences-verified knockout plasmids were electroporated into RN4220 integrated with prophage Tan2 using ELEPO21 electroporator (Nepa Gene, Japan).The following pulse parameters were used: Poring Pulse (voltage: 1800 V, pulse length: 2.5 ms, pulse interval: 50 ms, number of pulses: 1, Polarity: +); Transfer Pulse (voltage: 100 V, pulse length: 99 ms, pulse interval: 50 ms, number of pulses: 5, polarity: + / −) 32 .The transformants were grown on TSA supplemented with 5 µg/ml Tet at 30 ℃. Single crossover was achieved by growing the cells on TSA supplemented with 5 µg/ ml Tet at non-permissive temperature of 43 ℃.Finally, double crossover was performed by growing the cells on TSA supplemented with 5 µg/ml anhydrotetracycline at 37 ℃.The construction of S. aureus RN4220 ΦTan2ΔterL1/terS mutants was further confirmed by PCR and Sanger sequencing.
All primer and oligo DNA sequence used in this study were listed in Table S2, all plasmids used in this study were listed in Table S3 31,33 .
For the construction of phagemid-ΔLSAB(cut), phagemid-LSAB was linearized, with the exclusion of a 57 bp sequences at the 3ʹ end of terL, through PCR amplification using primers pLK19_1_F/pLK19_1_R.Subsequently, the amplified fragments were self-ligated using NEBuilder ® HiFi DNA Assembly to finally generate phagemid-ΔLSAB(cut).On the other hand, phagemid-LSAB(mosaic) was constructed by ligating two PCR-amplified fragments of phagemid-LSAB with a synthesized 57 bp mosaic sequence fragment, where 24 silent mutations (without amino acid substitution) were introduced, using NEBuilder ® HiFi DNA Assembly.
All constructed phagemids were subsequently transformed into E. coli DC10B and selected on LB agar supplemented with 10 µg/ml Cp.Successful transformation of each phagemid was validated by colony PCR.Following validation, the phagemids were extracted using FastGene ® plasmid mini kit (NIPPON Genetics, Japan).

Generation of phagemid-based staphylococcal AB-capsid
The constructed phagemids [phagemid-ΔLSAB(cut) and phagemid-LSAB(mosaic)] were individually transformed into host cell S. aureus RN4220 ΦTan2ΔterL/terS by electroporation using ELEPO21 electroporator (as previously described) 32 .Resulting transformants were recovered at a temperature permissive for plasmid replication (30 °C) for 5 h and then plated on TSA plates supplemented with Cp.Successful transformation of each phagemid was validated by performing colony PCR.Next, single PCR-confirmed colonies were inoculated in TSB containing Cp and cultured at 37 °C with shaking.Mitomycin C (FUJIFILM Wako Pure Chemicals, Japan) was added to a final concentration of 0.4 µg/ml when the bacterial culture reached OD600 of 0.5 to initiate the phage synthesis process, and subsequently, the phagemid DNAs were packaged, and AB-capsids were produced.Mitomycin C induction was carried out for 7 h at 30 °C with shaking at 80 rpm.After incubation, the culture was centrifuged and the supernatant removed.The pellet was then suspended in 1 ml of TSB containing Cp and incubated overnight at 30 °C.On the following day, the culture was added with another 1 ml of TSB containing Cp and centrifuged to pellet cell debris.The supernatant containing phagemid-based staphylococcal AB-capsids were passed through a 0.22 µm membrane filter and the AB-capsids solution was harvested.

Measurement of titers of phage and AB-capsid
The AB-capsid solution was serially tenfold diluted with SM buffer to a range of 10 −1 to 10 −7 .Meanwhile, an overnight culture of S. aureus strain RN4220, diluted 1:100 with TSB broth, was incubated with agitation at 37 °C until an OD 600 of approximately 0.5.Then, 110 µl of each dilution of AB-capsid solution was added to an equal volume of bacterial suspension, and the mixture was incubated at 37 °C for 20 min.After that, 100 µl of the mixture was added to 4 ml of soft top agar (TSB solution with 0.75% agarose) pre-warmed at 55 °C and overlaid on a TSA plate, and a Cp TSA plate, respectively.The solidified plates were incubated at 37 °C overnight.The colonies grown on the Cp TSA plate were counted to calculate the transduced colony-forming units, TFU/ml; whereas the plaques formed on the drug-free TSA plate were counted to calculate the plaque-forming units, PFU/ml.All assays were repeated three times (n = 3).

Spacers insertion for targeted bacterial killing
The CRISPR-Cas13a system cloned on phagemid-LSAB(mosaic) was designed to target nine drug-resistant genes: aadD, aph(3ʹ), aac(6ʹ), ermB, fosB, fusC, mphC, mecA, and tetK.The 25 bp long spacer sequences targeting each of the above genes, and a non-targeting spacer (irrelevant sequence) included as a control, were selected.A total of 10 types of 85-mer oligo DNAs, comprising a 25-mer spacer sequence (crRNA) complementary to the conserved region of each target gene, flanked upstream and downstream each with 30-mer nucleotides corresponding to the consensus sequences at the point of insertion on the phagemid, were custom-designed.After that, phagemid-LSAB(mosaic) was treated with the restriction enzyme BsaI-HF, purified with gel electrophoresis, and subsequently ligated with the 85-mer oligo DNAs using NEBuilder ® HiFi DNA Assembly to

Figure 2 .
Figure 2. Deletion of homologous sequences existing between prophage and phagemid.(A) Schematic illustration of the packaging genes carried by phagemid-LSAB and the prophage genome on the host strain RN4220 ΦTan2ΔterL/terS .The upper panel displays the host cell RN4220 ΦTan2ΔterL/terS used in our previous study (manuscript in preparation), while the lower panel shows the host cell RN4220 ΦTan2ΔterL1/terS with the deletion of a 57 bp homologous region used in the current study.The blue arrows indicate the packaging genes of the phagemid-LSAB.(B) Quantification of transduced colony-forming units (TFU) of AB-capsids and plaqueforming units (PFU) of wild-type phage packaged using both RN4220 ΦTan2ΔterL/terS and RN4220 ΦTan2ΔterL1/terS host cells transformed with phagemid-LSAB (n = 3).Red bars represent log TFU/ml, and blue bars represent log PFU/ml.Error bars represent standard deviations.N.D. not detected.

Figure 3 .
Figure 3. Homologous sequence modification and prevention of phage contamination.(A) Introduction of 24 silent mutations specifically into the packaging site of phagemid-LSAB(mosaic) to eliminate sequence homology.The upper panels show the packaging genes present on the three phagemids and the prophage of host RN4220 ΦTan2ΔterL/terS .The lower panel displays the sequence alignment of the 57 bp homologous regions of phagemid-LSAB and phagemid-LSAB(mosaic), where the silent mutations were introduced.The light red area indicates the 57 bp homologous regions.(B) Quantification of transduced colony-forming units (TFU) of AB-capsids and plaque-forming units (PFU) of wild-type phage packaged using host cell RN4220 Φtan2ΔterL/terS transformed with different phagemids (n = 3).Error bars represent standard deviations.N.D.; not detected.Student's t-test was performed to determine the significant difference between AB-capsids titer produced using phagemid-LSAB or phagemid-LSAB(mosaic).ns, not significant.(C) Representative agar plates with bacterial colonies formed by AB-capsids transduction (upper) and plaques formed by contaminated wild-type phages (lower).

Figure 4 .
Figure 4. Sequence-specific bacterial killing by AB-capsids generated using modified phagemid-LSAB(mosaic).(A) Schematic representation of the experimental flow for evaluating the bacterial killing activity of AB-capsids generated using phagemid-LSAB(mosaic) in this study.A tenfold serial dilution of AB-capsids was mixed with bacterial cultures of strains carrying target genes, and then 2.5 µl of the mixtures were spotted on TSB agar plates containing chloramphenicol and tetracycline to visualize bactericidal activity.(B) Sequences-specific bacterial killing of the AB-capsids was evaluated using target gene overexpressed transformants of S. aureus RN4220.The genes overexpressed in RN4220 are listed in the left columns, and the presence or absence of the target gene in the bacterium are shown with ( +) and ( −), respectively.(C,D) Sequence-specific bacterial killing of the AB-capsids against clinical isolates of methicillin-resistant S. aureus (MRSA).Besides mecA, JMUB1278 carries aac(6ʹ) associated with gentamicin and tobramycin resistance (aminoglycoside antibiotics); and JMUB3007 carries aadD that is associated with resistance to streptomycin (aminoglycoside antibiotics).Antibiotic resistance genes present in clinical isolates are highlighted in red font.Each gene listed on the left plane represents the different resistant gene-targeting AB-capsids used, with Non-T representing the nontargeting AB-capsid that lack spacers as control. https://doi.org/10.1038/s41598-024-67193-5